Kinetics of methyl group transfer between the Ni-Fe-S-containing acetyl-CoA synthase (ACS) and the corrinoid protein (CoFeSP) from Clostridium thermoaceticum were investigated using the stopped-flow method at 390 nm. Rates of the reaction CH3-Co3+FeSP + ACS(red);= Co1+FeSP + CH3-ACS(ox)., in both forward and reverse directions were determined using various protein and reductant concentrations. Tl3+-citrate, dithionite, and CO were used to reductively activate ACS (forming ACS(red)). The simplest mechanism that adequately fit the data involved formation of a [CH3-Co3+FeSP]:[ACS(red)] complex, methyl group transfer (forming [Co1+FeSP]:[CH3-ACS(ox)]), product dissociation (forming Co1+FeSP + CH3-ACS(ox)), and CO binding yielding a nonproductive enzyme state (ACS(red) -4- CO = ACS(red)-CO). Best-fit rate constants were obtained. CO inhibited methyl group transfer by binding ACS(red) in accordance with K-D = 180 +/- 90 muM. Fits were unimproved when > 1 CO was assumed to bind. Tl(3+)citrate and dithionite inhibited the reverse methyl group transfer reaction, probably by reducing the D-site of CH3-ACS(ox). This redox site is oxidized by 2e(-) when the methyl cation is transferred from CH3-Co3+FeSP to ACS(red), and is reduced during the reverse reaction. Best-fit K-D values for pre- and post-methyl-transfer complexes were 0.12 +/- 0.06 and 0.3 +/- 0.2 muM, respectively. Intracomplex methyl group transfer was reversible with K-eq = 2.3 +/- 0.9 (k(f)/k(f) = 6.9 s(-1)/3.0 s(-1)). The nucleophilicity of the {Ni2+Dred} unit appears comparable to that of Co1+ cobalamins. Reduction of the D-site may cause the Ni2+ of the A-cluster to behave like the Ni of an organometallic Ni-o complex.